肺炎链球菌表面抗原PsaA-PspA融合蛋白双抗夹心ELISA定量检测方法的建立及验证  

Development and verification of a double antibody sandwich ELISA for quantitative determination of PsaA-PspA fusion protein of Streptococcus pneumoniae surface antigen

作　　者：孙青 赵兴红 吴永革 谷铁军 SUN Qing;ZHAO Xing-hong;WU Yong-ge;GU Tie-jun(National Engineering Laboratory for AIDS Vaccine,Jilin University,Changchun 130012,Jilin Province,China)

机构地区：[1]吉林大学艾滋病疫苗国家工程实验室,吉林长春130012

出　　处：《中国生物制品学杂志》2018年第8期892-895,901,共5页Chinese Journal of Biologicals

摘　　要：目的建立快速测定肺炎链球菌表面抗原PsaA-PspA融合蛋白含量的双抗夹心ELISA方法,并进行验证。方法制备PsaA-PspA抗原,确定PsaA单抗和PsaA-PspA兔多抗的最佳工作浓度,建立双抗夹心ELISA方法。对方法的抗原特异性、精密性和准确性进行验证,并分析变性抗原对测定的影响。结果单抗和兔多抗最佳稀释倍数为1∶500和1∶10 000。建立的双抗夹心ELISA法检测PsaA-PspA抗原的浓度范围为70~560 ng/m L,R^2为0.993 8;该方法不适用于其他抗原的测定,但可用于鉴定抗原是否变性;该方法检测不同批次PsaA-PspA抗原的批内平均变异系数(CV)为4.9%,批间平均CV为19%,平均回收率为88.98%。结论建立的双抗夹心ELISA方法特异性、精密性和准确性良好,可用于PsaA-PspA融合蛋白浓度的检测,且可检测抗原是否变性。Objective To develop and verify a double antibody sandwich ELISA method for rapid detection of pheumococcal surface antigen PsaA-PspA fusion protein. Methods PsaA-PspA antigen was prepared,and the working concentrations of PsaA monoclonal antibody and rabbit anti-PsaA-PspA polyclonal antibody were optimized,based on which a double antibody sandwich ELISA method was developed and verified for specificity,precision and accuracy. The effect of denatured antigen on determination result was analyzed. Results The optimal dilutions of monoclonal antibody and rabbit polyclonal antibody were 1 ∶ 500 and 1 ∶ 10 000 respectively. The concentration range of PsaA-PspA for detection by the developed method was 70 - 560 ng/m L,with a R-2 value of 0. 993 8. The method was unsuitable for determination of other antigens,while was suitable for identifying whether the antigen was de-naturalized. The mean CV of determination results of PsaA-PspA of various batches by the method was 4. 9% in intra-assay,and 19% in inter-assay,while the mean recovery rate was 88. 98%. Conclusion The developed double antibody sandwich ELISA method showed high specificity, precision and accuracy, which might be used for determination of PsaA-PspA fusion protein and identifying whether PsaA-PspA was de-naturalized.

关 键 词：肺炎链球菌 表面抗原 PsaA-PspA融合蛋白 酶联免疫吸附测定 

分 类 号：R378.14[医药卫生—病原生物学] R392-33[医药卫生—基础医学]